PROJECT SUMMARY/ABSTRACT The intestinal tract mucosa in persons with inflammatory bowel disease and systemic immunosuppression is the major site of severe cytomegalovirus (CMV)-induced inflammation. Intestinal macrophages play a central role in mediating this inflammation, although intestinal macrophages are profoundly non-inflammatory in normal mucosa. To explain this fundamental paradox in which macrophages in normal mucosa are non-inflammatory but in CMV mucosal disease are potently inflammatory, we will elucidate the mechanism by which CMV, a dsDNA virus that persistently infects half the U.S. population and resides predominantly in blood monocytes, causes intestinal macrophages to become inflammatory. We have shown that (i) circulating blood monocytes are the exclusive source of intestinal macrophages, (ii) the inability of intestinal macrophages to mount inflammatory responses is due to stromal TGF-? induction of Smad pathway signaling, which leads to suppression of NF-?B, (iii) CMV infection of blood monocytes enhances monocyte inflammatory responses, and (iv) CMV infection of primary intestinal macrophages does not induce inflammatory function. However, our Preliminary Results show that CMV infection confers upon blood monocytes the ability to resist stromal TGF-?-induced differentiation into non-inflammatory macrophages, causing the mucosalized macrophages to become inflammatory. Therefore, we hypothesize that CMV infection of blood monocytes hijacks the Smad signaling pathway, thereby blocking stromal TGF-?- mediated inactivation of NF-?B in infected monocytes when the cells enter the mucosa, resulting in the differentiation of pro-inflammatory intestinal macrophages. Using our unique system in which blood monocytes are differentiated in vitro by stromal TGF-? into cells with the phenotype and function of primary intestinal macrophages (?mucosalized macrophages?) and CMV-infected DSS and TNBS mouse models, we will address this hypothesis with three Specific Aims: Specific Aim 1: Determine whether CMV infection causes blood monocytes to retain their inflammatory profile in the presence of TGF-?-containing S-CM in vitro. Specific Aim 2: Determine the role of Smad signaling in mouse CMV (MCMV)-enhanced colitis in the DSS and TNBS models of mucosal inflammation in vivo. Specific Aim 3: Determine whether CMV regulation of miR-146a, which mediates LPS tolerance, is involved in reversing S-CM down-regulation of monocyte inflammatory function. Addressing these fundamental scientific issues, we seek to uncover novel signaling targets to interdict inflammatory pathways in CMV mucosal disease.